Epidermal growth factor (EGF)-receptor signalling is needed for murine beta cell mass expansion in response to high-fat diet and pregnancy but not after pancreatic duct ligation.

AIMS/HYPOTHESIS: Epidermal growth factor receptor (EGFR) signalling is essential for the proper fetal development of pancreatic islets and in the postnatal formation of an adequate beta cell mass. In this study we investigated the role of EGFR signalling in the physiological states of beta cell mass expansion in adults during metabolic syndrome and pregnancy, as well as in regeneration after pancreatic duct ligation. METHODS: Heterozygous Pdx1-EGFR-dominant-negative (E1-DN) mice, which have a kinase-negative EGFR under the Pdx1 promoter, and wild-type mice were both subjected to a high-fat diet, pregnancy and pancreatic duct ligation. RESULTS: The beta cell mass of wild-type mice fed the high-fat diet increased by 70% and the mice remained normoglycaemic; the E1-DN mice became diabetic and failed to show any compensatory beta cell mass expansion. Similarly, pregnant wild-type mice had four times more proliferating beta cells and a 75% increase in beta cell mass at mid-gestation, in contrast to the pregnant E1-DN mice, which did not show any significant beta cell compensation and were hyperglycaemic in an intraperitoneal glucose tolerance test. However, after pancreatic duct ligation, both the wild-type and E1-DN mice showed similar expression of Ngn3 (also known as Neurog3) and beta cell proliferation increased to a similar level in the ligated part of pancreas. CONCLUSIONS/INTERPRETATIONS: EGFR signalling is essential in beta cell mass expansion during a high-fat diet and pregnancy where replication is the primary mechanism for compensatory beta cell mass expansion. In contrast, EGFR signalling appears not to be crucial to increased beta cell proliferation after pancreatic duct ligation.

Effects of immunosuppressive drugs on in vitro neogenesis of human islets: mycophenolate mofetil inhibits the proliferation of ductal cells.

Assuming that neogenesis contributes to long-term function of islet grafts, it is important to study the effects of immunosuppressive drugs on precursor cell proliferation and differentiation. We examined the effects of low-dose immunosuppressive drugs on these processes in vitro. Immunosuppressive drugs, including sirolimus, tacrolimus, mycophenolate mofetil (MMF), daclizumab and their combinations were tested in parallel culture wells through either the expansion phase (5-7 days) or the entire culture period (4-5 weeks). MMF, alone or in combination with sirolimus or tacrolimus, severely hampered duct-cell proliferation by 8-fold during the expansion period, and significantly reduced the total DNA content by about 40% after 5-week culture. After 4-5 week exposure to different drugs, only sirolimus and daclizumab showed no adverse effects on insulin content, whereas significant reductions of 30-60% in insulin content were seen in all other experimental groups. Only tacrolimus decreased the insulin content per DNA, as well as the proportion of insulin-positive cells. In conclusion, MMF has a potent inhibitory effect on neogenesis primarily through an antiproliferative effect on the precursors, whereas tacrolimus mainly affects beta-cell differentiation. Sirolimus and daclizumab have no adverse effects on these parameters. The immunosuppressive protocol may be an important determinant of long-term clinical islet graft function.

In vitro neogenesis of human islets reflects the plasticity of differentiated human pancreatic cells.

AIMS/HYPOTHESIS: The neogenesis of islets from cultured human adult pancreatic tissue has been reported. The islet progenitors have been thought to be ductal cells. Since previous experiments have been 'contaminated' by a number of pre-existing islet cells, we examined their involvement in islet cell neogenesis. METHODS: Fresh human pancreatic cells with different purities of islet cells were grown in monolayer culture and labelled with bromodeoxyuridine. Transitional cells were analysed by double immunofluorescence staining. For purified ductal cell culture, pre-existing islets were eliminated on a magnetic cell separation system. RESULTS: We confirmed that less than 1% of the endocrine cells proliferated, mainly during the first 48 h of culture. However, a 10-fold larger proportion of the cells acquired a transitional phenotype by starting to coexpress the ductal marker cytokeratin 19 (CK19). These cells represented more than 10% of all endocrine cells after 1 day in culture, and 6% at 5 days of culture. Using magnetic cell sorting, we eliminated cells expressing neural cell adhesion molecule (N-CAM), after which we obtained 99.7% pure non-endocrine CK19-rich cell populations. These cell populations could be expanded in vitro. However, their endocrine differentiation capacity was severely reduced as compared with the original mixed cell cultures. CONCLUSIONS/INTERPRETATION: These results suggest that islet neogenesis in this culture system at least partly represents the de-differentiation of islet cells into a duct-cell-like phenotype, with further re-differentiation in appropriate conditions. The plasticity of differentiated human pancreatic cell types may thus be an important mechanism of human pancreas regeneration.

Stimulated endocrine cell proliferation and differentiation in transplanted human pancreatic islets: effects of the ob gene and compensatory growth of the implantation organ.

Neogenesis is crucial for the maintenance of beta-cell mass in the human pancreas and possibly for the outcome of clinical islet transplantation. To date, no studies have reported a stimulation of human beta-cell neogenesis in vivo. Therefore, we investigated whether human alpha-, beta-, and duct cell growth can be stimulated when human islets are xenotransplanted to obese hyperglycemic-hyperinsulinemic ob/ob mice immunosuppressed with anti-lymphocyte serum. Moreover, we wanted to study whether beta-cell growth and duct-to-beta-cell differentiation were induced in the hepatocyte growth factor (HGF)-dependent compensatory kidney growth model. For that purpose, we evaluated human islets grafted to nude (nu/nu) mice before uninephrectomy of the contralateral kidney for DNA-synthesis and duct cell expression of the beta-cell-specific transcription factor Nkx 6.1 as an estimate of differentiation. Human islet grafts were well preserved after 2 weeks when transplanted to ob/ob mice during anti-lymphocyte immunosuppression. Both human beta-cells (P < 0.01) and duct cells (P < 0.001) were growth stimulated when islets were transplanted to ob/ob mice. We also observed a correlation between increased duct cell proliferation and increased organ donor age (P = 0.02). Moreover, duct (P < 0.05) and beta-cell (P < 0.05) proliferation, as well as duct cell Nkx 6.1 expression (P < 0.05), were enhanced by the compensatory kidney growth after uninephrectomy. We conclude that it is possible to stimulate human beta-cell neogenesis in vivo, provided that the recipient carries certain growth-stimulatory traits. Furthermore, it seems that duct cell proliferation increases with increasing organ donor age. Altogether, these data and previous results from our laboratory suggest that human beta-cell neogenesis becomes more dependent on differentiation and less dependent on proliferation with increasing age.

cDNA cloning, expression studies and chromosome mapping of human type I serine/threonine kinase receptor ALK7 (ACVR1C).

Transforming growth factor-beta (TGF-beta) superfamily related growth factors signal by binding to transmembrane type I and type II receptor serine/threonine kinases (RSTK), which phosphorylate intracellular Smad transcription factors in response to ligand binding. Here we describe the cloning of the human type I RSTK activin receptor-like kinase 7 (ALK7), an orthologue of the previously identified rat ALK7. Nodal, a TGF-beta member expressed during embryonic development and implicated in developmental events like mesoderm formation and left-right axis specification, was recently shown to signal through ALK7. We found ALK7 mRNA to be most abundantly expressed in human brain, pancreas and colon. A cDNA encoding the open reading frame of ALK7 was obtained from a human brain cDNA library. Furthermore, a P1 artificial chromosome (PAC) clone containing the human ALK7 gene was isolated and fluorescent in situ hybridization (FISH) on metaphase chromosomes identified the gene locus as chromosome 2q24.1-->q3. To test the functionality of the ALK7 signaling, we generated recombinant adenoviruses containing a constitutively active form of ALK7 (Ad-caALK7), which is capable of activating downstream targets in a ligand independent manner. Infection with Ad-caALK7 of MIN6 insulinoma cells, in which ALK7 has previously been shown to be endogenously expressed, led to a marked increase in the phosphorylation of Smad2, a signaling molecule also used by TGF-betas and activins.

Impaired migration and delayed differentiation of pancreatic islet cells in mice lacking EGF-receptors.

Pancreatic acini and islets are believed to differentiate from common ductal precursors through a process requiring various growth factors. Epidermal growth factor receptor (EGF-R) is expressed throughout the developing pancreas. We have analyzed here the pancreatic phenotype of EGF-R deficient (-/-) mice, which generally die from epithelial immaturity within the first postnatal week. The pancreata appeared macroscopically normal. The most striking feature of the EGF-R (-/-) islets was that instead of forming circular clusters, the islet cells were mainly located in streak-like structures directly associated with pancreatic ducts. Based on BrdU-labelling, proliferation of the neonatal EGF-R (-/-) beta-cells was significantly reduced (2.6+/-0.4 versus 5.8+/-0.9%, P<0.01) and the difference persisted even at 7-11 days of age. Analysis of embryonic pancreata revealed impaired branching morphogenesis and delayed islet cell differentiation in the EGF-R (-/-) mice. Islet development was analyzed further in organ cultures of E12.5 pancreata. The proportion of insulin-positive cells was significantly lower in the EGF-R (-/-) explants (27+/-6 versus 48+/-8%, P<0.01), indicating delayed differentiation of the beta cells. Branching of the epithelium into ducts was also impaired. Matrix metalloproteinase (MMP-2 and MMP-9) activity was reduced 20% in EGF-R (-/-) late-gestation pancreata, as measured by gelatinase assays. Furthermore, the levels of secreted plasminogen activator inhibitor-1 (PAI-1) were markedly higher, while no apparent differences were seen in the levels of active uPA and tPa between EGF-R (-/-) and wild-type pancreata. Our findings suggest that the perturbation of EGF-R-mediated signalling can lead to a generalized proliferation defect of the pancreatic epithelia associated with a delay in beta cell development and disturbed migration of the developing islet cells as they differentiate from their precursors. Upregulated PAI-1 production and decreased gelatinolytic activity correlated to this migration defect. An intact EGF-R pathway appears to be a prerequisite for normal pancreatic development.

Mechanisms of coxsackievirus-induced damage to human pancreatic beta-cells.

Enteroviruses may be involved in the pathogenesis of insulin-dependent diabetes mellitus, either through direct beta-cell infection or as triggers of autoimmunity. In the present study we investigated the patterns of infection in adult human islet cell preparations (consisting of 56+/-14% beta-cells) by several coxsackieviruses. The cells were infected with prototype strains of coxsackievirus B (CBV) 3, 4, and 5 as well as coxsackievirus A9 (CAV-9). The previously characterized diabetogenic strain of coxsackievirus B4 (CBV-4-E2) was used as a reference. All viruses replicated well in beta-cells, but only CBVs caused cell death. One week after infection, the insulin response of the beta-cells to glucose or glucose plus theophylline was most severely impaired by CBV-3 and CBV-5 infections. CBV-4 also caused significant functional impairment, whereas CAV-9-infected cells responded like uninfected controls. After 2 days of infection, about 40% of CBV-5-infected cells had undergone morphological changes characteristic of pyknosis, i.e. highly distorted nuclei with condensed but intact chromatin. Both mitochondria and plasma membrane were intact in these cells. DNA fragmentation was found in 5.9+/-1.1% of CBV-5-infected beta-cell nuclei (2.1+/-0.3% in controls; P<0.01). CAV-9 infection did not induce DNA fragmentation. One week after infection the majority of infected cells showed characteristics of secondary necrosis. Medium nitrite and inducible nitric oxide synthase messenger ribonucleic acid levels were not significantly up-regulated by CBV infection. These results suggest that several enteroviruses may infect human beta-cells. The infection may result in functional impairment or death of the beta-cell or may have no apparent immediate adverse effects, as shown here for CAV-9. Coxsackie B viruses cause functional impairment and beta-cell death characterized by nuclear pyknosis. Apoptosis appears to play a minor role during a productive CBV infection in beta-cells.

Differentiation and maturation of porcine fetal islet cells in vitro and after transplantation.

BACKGROUND: Porcine fetal pancreas is a potential source of beta cells for transplantation. The immaturity of the cells is a problem. We have defined the optimal conditions for in vitro propagation of this tissue before transplantation. METHODS: Porcine fetal pancreas tissue was obtained for tissue culture at various stages of development. Serum-containing and serum-free media and a variety of potential differentiation factors were tested. In vitro, the numbers of endocrine islet cells and their proliferation were quantified and functional maturity of the beta cells was assessed by perifusion. Growth and maturation of the cells was assessed 3 months after transplantation into nude mice. RESULTS: Highest beta cell mass was obtained from end-gestational, as compared with early fetal or neonatal, pancreas. Nicotinamide and sodium butyrate effectively increased the insulin content and the number of endocrine cells in culture. In combination, these factors led up to a 90-fold increase in the insulin content of islet-like cell clusters (ICC) as compared with untreated controls. However, a high level of cell death through apoptosis was observed in these maximally stimulated endocrine cells, and they did not survive as grafts when transplanted into nude mice. Instead, a serum-free culture medium containing 10 mM nicotinamide and 0.1 mM isobutylmethylxanthine was found to support both differentiation and proliferation of endocrine cells as loose ICCs. Insulin release from these ICCs was sensitive to glucose. When transplanted under the kidney capsule of normoglycemic nude mice, a high level of beta cell differentiation and function was evident only in the ICCs cultured in the serum-free medium, and in freshly isolated ICCs. When transplanted to hyperglycemic nude recipients, the cells cultured in serum-free medium for 3 weeks reversed hyperglycemia more consistently and rapidly than freshly isolated ICCs. CONCLUSIONS: Optimal maturation of porcine fetal pancreatic cells is obtained in serum-free medium supplemented with nicotinamide. Butyrate is a potent stimulus for beta cell differentiation but leads to increased apoptotic cell death.

Culture of adult human islet preparations with hepatocyte growth factor and 804G matrix is mitogenic for duct cells but not for beta-cells.

It has recently been reported that human adult beta-cells proliferate during culture on an extracellular matrix prepared from rat 804G cells and in the presence of hepatocyte growth factor (HGF). The present study compares the mitogenic effect of this condition on human beta-cells and on neighboring non-endocrine duct cells. Islet cell-enriched fractions were prepared from adult human organ donors and cultured in suspension or on 804G matrix, with or without HGF. The combination of 804G matrix and HGF increased the number of 5-bromo-2'-deoxyuridine-positive (BrdU+) cells within 48 h reaching a maximum after 4 days. In sections, virtually all BrdU+ cells were negative for insulin or glucagon and for preproinsulin mRNA but expressed the ductal cell markers cytokeratin 19 and 7, carbonic anhydrase-II, and carbohydrate antigen 19-9. After 4 days of culture, the cytokeratin 19+ ductal cells exhibited a BrdU-labeling index of 30% (P < 0.01 vs. 2% without HGF and matrix), whereas <0.1% of insulin-positive and <1% of glucagon-positive cells were labeled. Formation of bilayers with ductal cells covering the endocrine cells may cause erroneous interpretation on double positivity in unsectioned tissue. It is concluded that culture of human islet cell preparations with HGF and 804G matrix stimulates the proliferation of the duct cells but not of the underlying beta-cells.

Hyperlipidemia accelerates allograft arteriosclerosis (chronic rejection) in the rat.

The relevance of hyperlipidemia in allograft arteriosclerosis (chronic rejection) is controversial. Isolated hypercholesterolemia induced with cholesterol-cholic acid-diet (CC-diet) or hypertriglyceridemia induced with glycerol-diet (G-diet) had no or only a protective effect on aortic allograft arteriosclerosis in the rat. Combined hyperlipidemia with both diets (CC+G-diet) enhanced allograft arteriosclerosis by doubling intimal thickness and cellularity (P < .05) but had no effect on host arteries. Compared with normolipidemic controls, the CC+G-diet increased the total serum cholesterol concentration 4.8-fold (P < .05). Levels of VLDL2 and IDL increased 4.8- and 18.1-fold (P < .05), and their composition changed from triglyceride-rich to cholesterol-rich lipoproteins in an atherogenic direction. The CC+G-diet had no effect on the structure of inflammation in the vascular wall. Instead, significant lipid deposits were observed, and the expression of epidermal growth factor and insulin-like growth factor-1 was significantly elevated in the vascular wall. Thus, elevations in VLDL and IDL lipoprotein levels and their cholesterol content associate with the generation of allograft arteriosclerosis in rats. Deposition of lipids in the vascular wall seems to induce local synthesis of certain growth factors, which ultimately leads to the induction of smooth muscle cell replication.

Direct induction of class II molecules by cytomegalovirus in rat heart microvascular endothelial cells is inhibited by ganciclovir (DHPG).

It has been demonstrated both in vivo and in vitro that cytomegalovirus regulates not only MHC class I but also class II antigen expression on vascular endothelial cells. The CMV-linked MHC induction is believed to be involved in acute rejection mechanisms and chronic vasculopathy after transplantation. In this study, we have investigated the effect of 9-(1,3-dihydroxy-2-propoxymethyl) guanine (DHPG; ganciclovir) on CMV-induced class II expression in cultured rat heart microvascular endothelial cells. Two sets of cultured endothelial cells were infected with rat CMV. One of the sets was treated with various concentrations of DHPG and the other set was left untreated. MHC class II antigen expression on the cells was demonstrated by mAbs, by immunoperoxidase (IP) technique, and by FACS. Class II expression was maximal on CMV-infected cells 4-7 days after infection when 77.5 +/- 14.5% of the endothelial cells expressed class II in IP staining. DHPG inhibited the induction of class II, but the inhibitory effect was dependent upon the drug concentration. With increasing concentrations of DHPG (0, 1, 10, 100, and 1000 micrograms/ml), as demonstrated by IP staining (surface and intracellular expression), the frequency of class II-positive cells decreased from 77.5 +/- 14.5% to 58.0 +/- 15.0%, 36.0 +/- 23.0%, 3.0 +/- 3.0%, and 0 +/- 0%, respectively. By FACS (surface expression), the number of class II-positive cells remained somewhat lower, but decreased similarly with increasing concentrations of DHPG (from 37.8 +/- 1.1% to 6.2 +/- 3.1%) (surface expression). Also, the intensity of expression decreased concomitantly. These results suggest that DHPG inhibits CMV-induced class II expression via inhibition of CMV DNA polymerase.

CMV-induced class II antigen expression in various rat organs.

Cytomegalovirus (CMV) is thought to trigger acute or chronic allograft rejection by inducing the expression of MHC class II antigens in the graft. This induction may be mediated by gamma-interferon or directly by CMV. In this study, we have investigated which structures in the rat kidney, liver, and heart are responsive to CMV-induced class II expression in vivo. Rats were infected with rat CMV, the organs were harvested during the acute phase of infection, and the virus was demonstrated by culture from each organ. Direct CMV antigen detection was performed on frozen sections to demonstrate the detailed localization of CMV in the organs. In the kidney, CMV antigens were found in the vascular endothelium, in tubular cells, and scattered in the glomeruli. In the liver, the vascular structures and parenchyma contained CMV antigens. In the heart, CMV antigens were seen only in the capillary endothelium. Class II antigen expression was demonstrated by a monoclonal antibody and immunoperoxidase techniques. The induction of class II molecules was recorded in exactly the same cellular structures as those in which CMV antigens were detected.

Induction of class II molecules by cytomegalovirus in rat heart endothelial cells is inhibited by ganciclovir.

Cytomegalovirus (CMV) has been demonstrated to induce class II antigen expression in endothelial cells. To study whether ganciclovir (DHPG) has an effect on CMV-induced class II expression, cultured rat heart endothelial cells were infected with rat CMV (RCMV) and treated with different DHPG concentrations. Class II antigens in endothelial cells were detected by a monoclonal antibody and immunoperoxidase technique. Control cells did not express class II antigen, but during RCMV infection 92% of cells were class II-positive. DHPG treatment (1, 10, 100 and 1000 microg/ml) decreased RCMV-induced class II expression from 73% to 59%, 6% and 0%, respectively. As DHPG inhibits CMV DNA polymerase, our present results suggest that DHPG affects RCMV-induced class II expression via the inhibition of RCMV DNA replication.

Somatostatin analog lanreotide inhibits myocyte replication and several growth factors in allograft arteriosclerosis.

Chronic rejection is the most common reason for late loss of a transplant. The molecular mechanism of chronic rejection is not known and there is no treatment for this disorder. The characteristic histological feature in chronic rejection is increased smooth muscle cell replication in the vascular wall, leading to allograft arteriosclerosis. In this study we demonstrate that nonimmunosuppressed rat aortic allografts undergoing chronic rejection synthesize increased quantities of several smooth muscle cell growth-promoting substances in the vascular wall including interleukin-1, eicosanoids, and several peptide growth factors. Administration of a stable somatostatin analog lanreotide, BIM 23014, strongly inhibits myocyte proliferation in the allograft in vivo. It has no inhibitory effect on the proliferation of smooth muscle cells in vitro. Concomitantly, the locally produced peptide growth factors, i.e., epidermal growth factor, insulin-like growth factor 1, and BB-isomer of platelet-derived growth factor, but not other mediators of inflammation, are significantly reduced. The results suggest that growth factors are the main effector molecules leading to myocyte proliferation in allograft arteriosclerosis and that allograft arteriosclerosis (chronic rejection) may be specifically inhibited by lanreotide administration.

Interferon-gamma induces a phospholipase D-dependent release of arachidonic acid from endothelial cell membranes: a mechanism for protein kinase C activation.

Interferon-gamma (IFN-gamma) induces MHC class II expression on endothelial cells in a protein kinase C (PKC)-dependent manner. Here we show that IFN-gamma induces a sixfold arachidonic acid (AA) release from cultured rat microvascular endothelial cell membranes compared with non-treated cells. Since this result suggests that AA could act as a second messenger for IFN-gamma, we analysed its capacity to directly activate PKC. We have previously shown that IFN-gamma induces a transient, multiphasic activation of PKC via the action of the phospholipase D (PLD) pathway. Here we show that AA is able to activate PKC. In an attempt to characterize the source of the liberated AA after IFN-gamma induction in endothelial cells we used a panel of enzyme inhibitors. The IFN-gamma-induced release of AA could not be modified by interfering either with the phospholipase A2 (PLA2) pathway using bromophenacyl bromide (BPB), or with the phospholipase C (PLC) pathway using neomycin. The phosphatidic acid phosphatase (PAPase) inhibitor propranolol, inhibiting the generation of diacylglycerol (DAG) and further AA from phosphatidic acid (PA), could totally down-regulate the IFN-gamma-induced release of AA. Since PA is produced solely by the action of PLD from phosphatidylcholine (PC) we conclude that the AA originated from the cell membrane-associated PC. In summary, we show here that IFN-gamma causes the liberation of cell membrane-associated, PC-linked AA. This AA could directly activate PKC in a similar multiphasic manner to IFN-gamma, suggesting that it is a true second messenger for IFN-gamma in cultured endothelial cells.